The long-term objective of this program is to understand regulatory and processing phenomena that affect the expression of tRNA genes in the differentiating prokaryote Bacillus subtilis. These objectives are related to broader questions on the physiology, evolution, and genetic regulation in gram-positive bacteria, some of which cause human disease, and on the involvement of alterations in tRNAs in regulatory phenomena accompanying virus infections, differentiation, and neoplasia. B. subtilis is significantly different from Escherichia coli with respect to these studies because B. subtilis undergoes a differentiation process and has highly clustered tRNA genes. As a representative of gram-positive prokaryotes, studies with B. subtilis broaden our view of what is typical of eubacteria. Of special interest in their transcriptional regulation are two rRNA-tRNA operons of B. subtilis that contain large clusters of tRNA genes, most of which are under the transcriptional control of multiple promoter elements. Promoters for two tRNA genes, a minor 5 S rRNA, and the dual promoters of 16 S rRNA will be studied in fusion to the lacZ gene in a single-copy integration vector. Expression in B. subtilis during development will be evaluated by measuring levels of lacZ mRNA. Putative regulatory proteins will be looked for using both a genetic and biochemical approach. The autoregulation of tRNA genes will also be examined. Experiments on processing will concentrate on the importance of the structure of the substrate for efficient processing by the catalytic RNA of B. subtilis RNase P. In addition to monomeric and multimeric substrates, mixed precursors with ribo- and deoxyribonucleotides will be used to test the importance of 2'-OHs and conformation in substrate-"enzyme" interactions. The effects of conformational changes in E. coli 4.5 S RNA and the viral tRNA-like pseudoknot of TYMV on kinetic parameters with the normal and altered substrates will be determined using catalytic RNAs from E. coli and B. subtilis. Kinetic parameters of a smaller version of the catalytic RNA of RNase P (mini-P) will be examined using various substrates to see how closely it maintains the functional domains of the full length RNA. The conformation of small substrates as well as mini-P will be analyzed with collaborators expert in RNA structural analyses.